Haemophilus influenzae is common cause of localized respiratory tract disease, including otitis media, sinusitis, bronchitis, and pneumonia. Less commonly, this organism causes serious systemic disease, such as meningitis, endocarditis, and septicemia. The initial step in the pathogenesis of H. influenzae disease involves colonization of the upper respiratory mucosa. We have identified a high-molecular-weight protein called Hia, which is present in nontypable (nonencapsulated) H. influenzae and promotes attachment to human epithelium. In addition, we have identified a homolog of Hia called Hsf, which is univerally present among encapsulated H. influenzae and also mediates in vitro adherence. Interestingly, Hia has a predicted molecular mass of approximately 114 kDa and is not detectable by coventional transmission electron microscopy, while Hsf has a predicted molecular mass of approximately 245 kDa and is associated with the presence of short, thin surface fibrils visible by negative staining electron microscopy. Based on our in vitro results, we speculate that Hia and Hsf are important colonization factors. In the present proposal, we plan to characterize the pathway by which Hia and Hsf are localized on the surface of the organism. In particular, we will define the structural features of these proteins that direct them to the periplasm and facilitate their translocation across the outer membrane. We will dissect the influence of an unusual N-terminal extremity, a C-terminal domain predicted to form a beta-barrel, and a putative ATP-binding motif. In additional studies, we will examine the architecture of Hia and will investigate the relationship between structure and adhesive activity, focusing in particular on the role of a predicted coiled coil motif. We will also determine whether Hia and Hsf function interchangeably in an encapsulated strain. From a practical perspective, the results of these experiments may facilitate efforts to develop a vaccine protective against non-type b H. influenzae and suggest targets for novel antimicrobials with activity against a broad range of gram-negative bacteria. More generally, they may provide fundamental insights into the biogenesis of non-pilus adhesins and the nature of the host-microbial relationship.